1. Field of the Invention
The present invention relates to a nucleic acid complex comprising a high molecular weight derivative of ε-poly-L-lysine and a nucleic acid, and a method of introducing a nucleic acid complex into a cell, comprising administering the nucleic acid complex to a cell.
2. Description of the Related Art
Conventionally, methods of introducing a nucleic acid such as a gene into a cell are broadly classified into: introduction methods using chemical or physical means (nonviral method) such as the calcium phosphate method, polymer method such as DEAE-dextran method, liposome method, electroporation method, and microinjection method; and biological methods (viral method) utilizing a viral vector such as adenovirus vector or retrovirus vector, all of which are widely utilized.
Meanwhile, the biological method utilizing the infectivity of a virus has the advantage of a high gene introduction and expression efficiency, but it is pointed out that it has problems relating to safety of the virus itself, such as immunogenicity, probability of recurrence of pathogenicity and cell canceration, and the accidents actually happened (Marshall, E., et al., Science, 286, 2244 (1999), and Hacein-Bey-Abina, S. et al., Science, 302, 415 (2003)).
On the other hand, great expectations for the nonviral methods have been raised, because of its advantages including lower risks for safety compared with the viral method, easiness in a procedure of introducing a nucleic acid, and availability of an inexpensive, large-scale preparation of an introduction agent. However, it is a significant issue to be improved that the method has lower introduction and expression efficiency than a viral vector. To increase the efficiency of the nonviral methods, it is required to overcome several problems such as cell selectivity, cell membrane-permeability, nuclease-resistance, nuclear entry efficiency, nucleic acid releasability, and the like. As in the viral method, the nonviral methods also have important issues of safety assurance such as decrease in toxicity to a living body or a cell.
The liposome method, which is one of the chemical methods, has advantages in that a lipid in a liposome membrane is easy to interact with a cell membrane or an intracellular organ and has low antigenecity because it is composed of the same lipids as a biomembrane in most cases. However, gene introduction by means of a liposome has a disadvantage in that it is hardly provided for a practical use owing to problems such as low gene expression efficiency and high cytotoxicity (Chassin, D. et al., Eur. J. Immunol., 29, 196 (1999), and Van Tendeloo, V. F. et al., Gene Ther., 5, 700 (1998)).
Examples of typical chemical means include the polymer method as well as the liposome method. The polymer method draws attention as a nonviral method that enables various functionalizations by chemical modification. Among polymeric substances, polyethyleneimine that is a cationic polymer has an excellent ability for DNA compaction, which gives high introduction efficiency. However, it has a disadvantage of extremely strong cytotoxicity. Therefore, to solve the problem relating to safety such as cytotoxicity, it is necessary to impart biodegradability or biocompatibility to polymers, and there has also been studied a technique using a naturally derived cationic polymer that inherently has biodegradability or biocompatibility. Among polysaccharides, α-glucan-based dextran and β-1,4-glucosamine-based chitosan derivatives have been studied. However, DEAE-dextran or the like has a problem of extremely strong cytotoxicity, and chitosan also has problems that conditions for forming a DNA complex are limited and that a large amount of DNA is required for a sufficient gene expression (MacLaughlin, F. C. et al., J. Controlled Rel., 56, 259 (1998), and Ishii, T. et al., Biochim. Biophys. Acta, 1514, 51(2001), and JP 2000-157270 A).
Also, cationic polypeptides have been examined as gene introduction agents, and in particular, many studies have been made on α-poly-L-lysine because it has ability for DNA compaction. However, α-poly-L-lysine is known to have problems such as an insufficient gene introduction activity, high aggregation of a DNA complex, and high cytotoxicity (Brown, M. D. et al., Bioconjug. Chem., 11, 880 (2000)).
Therefore, in order to introduce a nucleic acid into a cell, development of a nucleic acid introduction agent which can be prepared easily and inexpensively, and has low cytotoxicity, and has excellent nucleic acid introduction efficiency, and an introduction method using the same has been strongly desired.